So far, 67 intermediate filament (IF) genes have been identified in humans. Mutations in them are responsible for over 30 human diseases, yet the in vivo properties of IF proteins are poorly understood, due in part to functional redundancy and a lack of simple, single-cell genetic model systems for IF study. Our recent discovery of crescentin, a bacterial cytoskeletal element with a high degree of similarity with IF proteins, provides a highly tractable bacterial system as a tool for understanding IF function. The objective of this project is to elucidate the mechanism by which crescentin causes cell curvature in Caulobacter crescentus. Specifically, this will be accomplished using three approaches. 1) Construction of a large set of crescentin mutants and characterization of their properties both in vitro and in vivo will determine structural requirements for making a cytoskeletal element with IF properties. 2) Fluorescence microscopy techniques combined with quantitative immunoblotting will enable the analysis of crescentin structure, localization, and dynamics within cells. Changes occurring during the cell cycle will be determined with the use of synchronized cell cycle populations. Additionally, the effect of crescentin on the bacterial cell wall shape (analogous to the extracellular matrix of animal cells) will be analyzed by determining patterns of cell wall growth in the presence and absence of crescentin, and by rapidly disrupting crescentin structure within live cells to observe any immediate morphological changes. 3) The roles of the highly conserved molecular chaperone DnaK (Hsp70) and the actin homolog MreB in crescentin assembly and function will be determined by observing crescentin structure, synthesis and stability within cells using fluorescence microscopy and metabolic labeling under conditions of DnaK or MreB loss-of-function. Co-localization, pull- down and in vitro experiments will examine the interaction of DnaK and MreB with crescentin. RELEVANCE: Many human diseases, ranging from skin blistering and muscular dystrophy to lipodystrophy and premature aging, are caused by abnormalities in intermediate filament proteins. This study of crescentin, a bacterial version of an intermediate filament protein, is likely to provide insight into the way its human counterparts assemble and function within cells. Knowing the basis for intermediate filament function may reveal how intermediate filament-based diseases develop, suggesting treatment strategies. [unreadable] [unreadable] [unreadable]